Messages pass constantly from the extracellular world into the interior of the cell. Many of these messages must travel to the nucleus to affect cellular change. A necessary step is passage through elaborate, highly regulated nuclear gateways, the nuclear pore complexes. When this traffic is perturbed, cancer, developmental defects, or aberrant cellular function can ensue. The vertebrate nuclear pore is 120 million daltons in size with a complex structural architecture. Key among the subunits that make up this gateway is the Nup107-160 complex, the so-called linchpin of the nuclear pore. Analysis of the binding partners of this key subunit revealed to us the protein ELYS, a protein that specifically targets pore assembly to the surface of the chromatin. Without ELYS pores fail to form at the nucleus and instead form in cytoplasmic membrane stacks. Clearly, ELYS is of primary importance to our existence as eukaryotes. Without it, our genomes would have no communication with the outer world of the cell. The present grant has four major goals: (1) The first is to determine the molecular interactions of ELYS: its chromatin binding targets, its rules of engagement with the Nup107-160 complex, and its recruitment of vesicles containing key integral membrane pore proteins. This knowledge is key to understanding how these key gateways are formed. (2) A next goal is to determine the physical steps of nuclear pore assembly, with specific analysis of the fusion event that occurs between the inner and outer membrane of the nucleus to initiate pore formation. For this, we will use novel nuclear intermediates and assays that we have recently developed. (3) Our third goal will be to probe the mechanism of action of a negative regulator of both nuclear membrane formation and nuclear pore assembly, importin [unreadable]. We will specifically ask which of the steps of nuclear pore assembly identified above are targeted by its regulation. (4) Lastly, we will attack a new challenge, the role of the protein centrin which we have recently discovered to be associated with vertebrate nuclear pores. We find centrin mutants to block both mRNA and protein export from the nucleus. We will ask which of the export factors or pathways show interaction with centrin in order to define its role at the nuclear pore. Successful completion of these goals will provide essential insight into the assembly and function of the nuclear pore, vital gateway to the nucleus. PUBLIC HEALTH RELEVANCE: The genome of each of us is contained within a fortress, the nucleus, and can be accessed only through intricate gateways called nuclear pores. No signals can reach the genome, nor messages be sent from it except through these pores. Understanding how the gateways are built and how they function is essential not only to understanding how the genome can be reached, but also how it is protected.